Insight Into the Degradation Pathways of an AAV9.

The use of recombinant adeno-associated virus (AAV) vectors is a popular choice for in vivo gene therapy, with hundreds of ongoing clinical trials targeting various genetic diseases. However, due to limited material availability and the complexity of AAV structure, there is a critical lack of comprehensive studies on AAV degradation pathways. In this study, we intended to elucidate the degradation pathways for a model AAV9 with GFP as the transgene under relevant stressed conditions. We assessed a diverse set of critical quality attributes and examined the overall impact of various stresses on transgene expression. This assessment revealed various degradation mechanisms of AAV9 and demonstrated the potential risk of a base formulation in causing AAV9 instability and potency loss under thermal stress at 25 and 40 °C while maintaining stability under freeze-thaw stress, interfacial stress due to membrane filtration, and short-term storage of up to 4 weeks at 5 °C.

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Intranasal murine pneumonia virus-vectored SARS-CoV-2 vaccine induces mucosal and serum antibodies in macaques.

Next-generation SARS-CoV-2 vaccines are needed that induce systemic and mucosal immunity. Murine pneumonia virus (MPV), a murine homolog of respiratory syncytial virus, is attenuated by host-range restriction in nonhuman primates and has a tropism for the respiratory tract. We generated MPV vectors expressing the wild-type SARS-CoV-2 spike protein (MPV/S) or its prefusion-stabilized form (MPV/S-2P). Both vectors replicated similarly in cell culture and stably expressed S. However, only S-2P was associated with MPV particles. After intranasal/intratracheal immunization of rhesus macaques, MPV/S and MPV/S-2P replicated to low levels in the airways. Despite its low-level replication, MPV/S-2P induced high levels of mucosal and serum IgG and IgA to SARS-CoV-2 S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2.

Sarcoma Immunotherapy: Confronting Present Hurdles and Unveiling Upcoming Opportunities.

Sarcomas are rare and heterogeneous mesenchymal neoplasms originating from the bone or soft tissues, which pose significant treatment challenges. The current standard treatment for sarcomas consists of surgical resection, often combined with chemo- and radiotherapy; however, local recurrence and metastasis remain significant concerns. Although immunotherapy has demonstrated promise in improving long-term survival rates for certain cancers, sarcomas are generally considered to be relatively less immunogenic than other tumors, presenting substantial challenges for effective immunotherapy. In this review, we examine the possible opportunities for sarcoma immunotherapy, noting cancer testis antigens expressed in sarcomas. We then cover the current status of immunotherapies in sarcomas, including progress in cancer vaccines, immune checkpoint inhibitors, and adoptive cellular therapy and their potential in combating these tumors. Furthermore, we discuss the limitations of immunotherapies in sarcomas, including a low tumor mutation burden and immunosuppressive tumor microenvironment, and explore potential strategies to tackle the immunosuppressive barriers in therapeutic interventions, shedding light on the development of effective and personalized treatments for sarcomas. Overall, this review provides a comprehensive overview of the current status and potential of immunotherapies in sarcoma treatment, highlighting the challenges and opportunities for developing effective therapies to improve the outcomes of patients with these rare malignancies.

Plasma zinc, copper and serum ceruloplasmin levels of autism spectrum disorder children in Bangladesh.

Neural and cognitive processes require zinc and copper homeostasis and a normal zinc/copper ratio. Ceruloplasmin, an intrinsic antioxidant protein, maintains copper homeostasis, which might also influence autism spectrum disorder (ASD). ASD children are frequently reported with altered levels of these elements with wide geographical variations. This study evaluated any alteration in plasma zinc, copper, zinc/copper ratio and serum ceruloplasmin levels in Bangladeshi ASD children with respect to healthy controls. A cross-sectional study was conducted on 67 children aged 2 to 9 years of both sexes. Among them, 35 had ASD, while 32 were age, sex and body mass index (BMI) matched apparently healthy children. Plasma zinc and copper levels were estimated by the flame atomic absorption spectrophotometry method. Serum ceruloplasmin levels were estimated by the immunoturbidimetric method. Zinc and zinc/copper ratio in the 2-9 years old ASD children group were significantly lower (p=0.032 and p=0.002 respectively). On the other hand, copper (p=0.020) and ceruloplasmin (p = 0.045) levels were significantly higher than those of apparently healthy children. ASD was significantly associated with zinc deficiency (p=0.000) and copper toxicity (p=0.05). All children were again divided into 2-5 and 6-9 years age groups according to laboratory reference values for zinc and copper. Copper toxicity was significantly associated with ASD in the 2-5 years old age group (p=0.011), with a significant difference in plasma copper levels (p=0.009) and zinc/copper ratio (p=0.001) but not serum ceruloplasmin levels (p=0.110) compared to healthy controls. Serum ceruloplasmin was positively associated with plasma copper in ASD children of all age groups. This study shows that ASD in Bangladesh can be associated with low plasma zinc and high plasma copper and serum ceruloplasmin levels.

Tea tree oil, a vibrant source of neuroprotection via neuroinflammation inhibition: a critical insight into repurposing Melaleuca alternifolia by unfolding its characteristics. / èŒ¶æ ‘æ²¹æŠ‘åˆ¶ç¥žç»ç‚Žç—‡å¹¶æä¾›ç¥žç»ä¿æŠ¤­­æŽ¢ç´¢äº’花千层的潜力.

Over the past few decades, complementary and alternative treatments have become increasingly popular worldwide. The purported therapeutic characteristics of natural products have come under increased scrutiny both in vitro and in vivo as part of efforts to legitimize their usage. One such product is tea tree oil (TTO), a volatile essential oil primarily obtained from the native Australian plant, Melaleuca alternifolia, which has diverse traditional and industrial applications such as topical preparations for the treatment of skin infections. Its anti-inflammatory-linked immunomodulatory actions have also been reported. This systematic review focuses on the anti-inflammatory effects of TTO and its main components that have shown strong immunomodulatory potential. An extensive literature search was performed electronically for data curation on worldwide accepted scientific databases, such as Web of Science, Google Scholar, PubMed, ScienceDirect, Scopus, and esteemed publishers such as Elsevier, Springer, Frontiers, and Taylor & Francis. Considering that the majority of pharmacological studies were conducted on crude oils only, the extracted data were critically analyzed to gain further insight into the prospects of TTO being used as a neuroprotective agent by drug formulation or dietary supplement. In addition, the active constituents contributing to the activity of TTO have not been well justified, and the core mechanisms need to be unveiled especially for anti-inflammatory and immunomodulatory effects leading to neuroprotection. Therefore, this review attempts to correlate the anti-inflammatory and immunomodulatory activity of TTO with its neuroprotective mechanisms.

Intranasal immunization with avian paramyxovirus type 3 expressing SARS-CoV-2 spike protein protects hamsters against SARS-CoV-2.

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters.

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

Virtual screening of functional foods and dissecting their roles in modulating gene functions to support post COVID-19 complications.

COVID-19 has become the focal point since 2019 after the outbreak of coronavirus disease. Many drugs are being tested and used to treat coronavirus infections; different kinds of vaccines are also introduced as preventive measure. Alternative therapeutics are as well incorporated into the health guidelines of some countries. This research aimed to look into the underlying mechanisms of functional foods and how they may improve the long-term post COVID-19 cardiovascular, diabetic, and respiratory complications through their bioactive compounds. The potentiality of nine functional foods for post COVID-19 complications was investigated through computational approaches. A total of 266 bioactive compounds of these foods were searched via extensive literature reviewing. Three highly associated targets namely troponin I interacting kinase (TNNI3K), dipeptidyl peptidase 4 (DPP-4), and transforming growth factor beta 1 (TGF-ß1) were selected for cardiovascular, diabetes, and respiratory disorders, respectively, after COVID-19 infections. Best docked compounds were further analyzed by network pharmacological tools to explore their interactions with complication-related genes (MAPK1 and HSP90AA1 for cardiovascular, PPARG and TNF-alpha for diabetes, and AKT-1 for respiratory disorders). Seventy-one suggested compounds out of one-hundred and thirty-nine (139) docked compounds in network pharmacology recommended 169 Gene Ontology (GO) items and 99 Kyoto Encyclopedia of Genes and Genomes signaling pathways preferably AKT signaling pathway, MAPK signaling pathway, ACE2 receptor signaling pathway, insulin signaling pathway, and PPAR signaling pathway. Among the chosen functional foods, black cumin, fenugreek, garlic, ginger, turmeric, bitter melon, and Indian pennywort were found to modulate the actions. Results demonstrate that aforesaid functional foods have attenuating roles to manage post COVID-19 complications. PRACTICAL APPLICATIONS: Functional foods have been approaching a greater interest due to their medicinal uses other than gastronomic pleasure. Nine functional food resources have been used in this research for their traditional and ethnopharmacological uses, but their directive-role in modulating the genes involved in the management of post COVID-19 complications is inadequately studied and reported. Therefore, the foods types used in this research may be prioritized to be used as functional foods for ameliorating the major post COVID-19 complications through appropriate science.

Genetic polymorphisms in CDH1 and Exo1 genes elevate the prostate cancer risk in Bangladeshi population.

The polymorphisms of invasion suppressor gene CDH1 and DNA mismatch repair gene Exo1 have been reported to play critical role in the development, tumorigenesis, and progression of several kinds of cancers including prostate cancer. This study was designed to analyze the contribution of single-nucleotide polymorphisms of the CDH1 (-160C/A) and Exo1 (K589E) to prostate cancer susceptibility in Bangladeshi population. The study included 100 prostate cancer cases and age-matched 100 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to determine the genetic polymorphisms. A significant association was found between CDH1 -160C/A (rs16260) and Exo1 (rs1047840, K589E) polymorphisms and prostate cancer risk. In case of CDH1 -160C/A polymorphism, the frequencies of the three genotypes C/C,C/A, and A/A were 45%, 48%, and 7% in cases and 63%, 32%, and 5% in controls, respectively. The heterozygote C/A genotype and combined C/A + A/A genotypes showed 2.10-fold (odds ratio = 2.1000, 95% confidence interval = 1.2956-4.0905, p = 0.013) and 2.08-fold (odds ratio = 2.0811, 95% confidence interval = 1.1820-3.6641, p = 0.011) increased risk of prostate cancer, respectively, when compared with homozygous C/C genotypes. The variant A allele also was associated with increased risk of prostate cancer (odds ratio = 1.6901, 95% confidence interval = 1.0740-2.6597, p = 0.0233). In case of Exo1 (K589E) polymorphism, G/A heterozygote, A/A homozygote, and combined G/A + A/A genotypes were found to be associated with 2.30-, 4.85-, and 3.04-fold higher risk of prostate cancer, respectively (odds ratio = 2.3021, 95% confidence interval = 2.956-4.0905, p = 0.0031; odds ratio = 4.8462, 95% confidence interval = 1.0198-23.0284, p = 0.0291; OR = 3.0362, 95% confidence interval = 1.7054-5.4053, p = 0.0001, respectively). The "A" allele showed significant association with increased susceptibility (2.29-fold) to prostate cancer (odds ratio = 2.2955, 95% confidence interval = 1.4529-3.6270, p = 0.0004). Our results suggest that CDH1 -160C/A and Exo1 K589E polymorphisms are associated with increased susceptibility to prostate cancer in Bangladeshi population.

The bovine herpesvirus-1 major tegument protein, VP8, interacts with host HSP60 concomitant with deregulation of mitochondrial function.

The UL47 gene product, VP8, is a major tegument protein of BoHV-1. While VP8 is not essential for virus replication in cell culture, a UL47-deleted virus exhibits a smaller tegument structure and is avirulent in cattle. To obtain pure VP8 protein for structural analysis, we expressed a N-terminally truncated version of VP8 in Eschericia coli. However, the recombinant VP8 was consistently co-purified with a tightly associated bacterial protein; this protein was identified by mass spectrometry as GroEL, which has considerable homology with mammalian heat shock protein-60 (HSP60), thus suggesting a new role for VP8 in virus-host interaction. A physical interaction of HSP60 and VP8 in both VP8-transfected and BoHV-1-infected cells was demonstrated by immunoprecipitation. Analysis of different truncated VP8 constructs revealed that amino acids 259-482 and 632-741 are involved in binding to HSP60. Full-length VP8 and VP8 219-741 (containing both interacting domains, 259-482 and 632-741) co-localized with HSP60 and mitochondria. VP8 was localized in the mitochondria from 2 to 14 h post infection in BoHV-1-infected cells. The mitochondrial membrane potential was reduced in both VP8-transfected and BoHV-1-infected cells and was further diminished by overexpression of HSP60 in the presence of VP8. In addition, VP8 expression decreased the ATP concentration during transfection, as well as BoHV-1 infection. Thus, VP8 may play a role in the deregulation of mitochondrial function through interaction with HSP60. This is consistent with the fact that BoHV-1 infection is known to promote mitochondrial dysfunction.

The Major Tegument Protein of Bovine Herpesvirus 1, VP8, Interacts with DNA Damage Response Proteins and Induces Apoptosis.

VP8, the UL47 gene product in bovine herpesvirus-1 (BoHV-1), is a major tegument protein that is essential for virus replication in vivo The major DNA damage response protein, ataxia telangiectasia mutated (ATM), phosphorylates Nijmegen breakage syndrome (NBS1) and structural maintenance of chromosome-1 (SMC1) proteins during the DNA damage response. VP8 was found to interact with ATM and NBS1 during transfection and BoHV-1 infection. However, VP8 did not interfere with phosphorylation of ATM in transfected or BoHV-1-infected cells. In contrast, VP8 inhibited phosphorylation of both NBS1 and SMC1 in transfected cells, as well as in BoHV-1-infected cells, but not in cells infected with a VP8 deletion mutant (BoHV-1ΔUL47). Inhibition of NBS1 and SMC1 phosphorylation was observed at 4 h postinfection by nuclear VP8. Furthermore, UV light-induced cyclobutane pyrimidine dimer (CPD) repair was reduced in the presence of VP8, and VP8 in fact enhanced etoposide or UV-induced apoptosis. This suggests that VP8 blocks the ATM/NBS1/SMC1 pathway and inhibits DNA repair. VP8 induced apoptosis in VP8-transfected cells through caspase-3 activation. The fact that BoHV-1 is known to induce apoptosis through caspase-3 activation is in agreement with this observation. The role of VP8 was confirmed by the observation that BoHV-1 induced significantly more apoptosis than BoHV-1ΔUL47. These data reveal a potential role of VP8 in the modulation of the DNA damage response pathway and induction of apoptosis during BoHV-1 infection.IMPORTANCE To our knowledge, the effect of BoHV-1 infection on the DNA damage response has not been characterized. Since BoHV-1ΔUL47 was previously shown to be avirulent in vivo, VP8 is critical for the progression of viral infection. We demonstrated that VP8 interacts with DNA damage response proteins and disrupts the ATM-NBS1-SMC1 pathway by inhibiting phosphorylation of DNA repair proteins NBS1 and SMC1. Furthermore, interference of VP8 with DNA repair was correlated with decreased cell viability and increased DNA damage-induced apoptosis. These data show that BoHV-1 VP8 developed a novel strategy to interrupt the ATM signaling pathway and to promote apoptosis. These results further enhance our understanding of the functions of VP8 during BoHV-1 infection and provide an additional explanation for the reduced virulence of BoHV-1ΔUL47.

VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling.

UNLABELLED: The UL47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a UL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-ß) signaling by using an IFN-α/ß-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-ß signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-ß signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells. IMPORTANCE: Since VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication. Indeed, while nonessential in vitro, it is critical for BoHV-1 replication in vivo In this study, we determined that VP8 plays a role in downregulation of the antiviral host response by inhibiting IFN-ß signaling. VP8 interacted with and prevented nuclear accumulation of STAT1 at 2 h postinfection in the absence of de novo viral protein synthesis. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for this interaction. These results provide a new functional role for VP8 in BoHV-1 infection and a potential explanation for the lack of viral replication of the UL47 deletion mutant in cattle.

Regulation and function of phosphorylation on VP8, the major tegument protein of bovine herpesvirus 1.

UNLABELLED: The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S(16) is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S(32). CK2 has multiple active sites, among which T(107) appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8. IMPORTANCE: The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S(16) initiates further phosphorylation at S(32) by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T(107) having the highest level of phosphorylation. Evidence for a difference in the phosphorylation status of VP8 in host cells and mature virus is presented for the first time. Phosphorylation was found to be a critical modification, which enables VP8 to attract and to redistribute PML protein in the nucleus. This might promote viral replication through interference with a PML-mediated antiviral defense. This study provides new insights into the regulation of VP8 phosphorylation and suggests a novel, phosphorylation-dependent function for VP8 in the life cycle of BoHV-1, which is important in view of the fact that VP8 is essential for virus replication in vivo.

Interaction of VP8 with mRNAs of bovine herpesvirus-1.

The UL47 gene product, VP8, is one of the most abundant tegument proteins of bovine herpesvirus-1 (BoHV-1). Deletion of VP8 leads to impaired growth in tissue culture, and VP8 is indispensable for BHV-1 replication in cattle. To elucidate the biological functions of VP8, we explored its interaction with mRNAs of immediate early (bICP0), early (gB, gD) and late (gC) genes of BoHV-1. FLAG-tagged VP8 was pulled down from COS-7 cells co-transfected with plasmids encoding VP8 and either gB, gC, gD or bICP0. This was followed by RNA extraction, cDNA synthesis and qPCR, which demonstrated binding of VP8 to bICP0, gB, gC and gD mRNAs in the cytoplasm and nucleus. These results were supported by co-localization of VP8 with bICP0, gB, gC and gD mRNAs in the nucleus as determined by confocal microscopy. Amino acids 259-342, located in the conserved portion of UL47 homologues, were found to contain the RNA binding region on VP8. To further characterize these interactions, Northwestern blotting was performed by immobilizing VP8 on a nitrocellulose membrane followed by hybridization with in vitro transcribed bICP0 mRNA. The results demonstrated binding of VP8 to intron-less mRNA but not intron-containing mRNA of bICP0. In addition, the interaction of VP8 with bICP0 mRNA was confirmed in vitro by RNA electrophoretic mobility shift assay, which also showed that the zinc finger and acidic domains both interact with VP8. Based on these results, we concluded that VP8 binds to intron-less mRNAs of bICP0, gB, gC and gD.

Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity.

The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promising source of antifungal drug targets. Galactofuranose (Galf) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of the Aspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A. nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Galf biosynthesis proteins each caused similar hyphal morphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we generated ugeA, ugmA and ugtA strains controlled by the alcA(p) or niiA(p) regulatable promoters. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) and niiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Galf immunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Galf. None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p), OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lower caspofungin sensitivity. Galf appears to play an important role in A. nidulans growth and vigor.

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity.

Galactofuranose (Galf) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Galf from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Galf residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Galf transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. AfglfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Galf, which was restored in the AfglfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.